Aging is associated with an increase in bone marrow adipocytes and a relative deficit in osteoblasts. Thus, it has been proposed that with senescence, there is a shunting of the common marrow precursor cell away from the osteoblast and towards the adipocyte lineage. Since the increase in marrow adipocytes and relative deficit in bone formation occurs in both women and men, and men were not felt to become sex steroid deficient with aging, it was generally believed that the increase in bone marrow adipocytes with age was independent of sex steroids. However, considerable work by us and others has established that while men do not have the equivalent of a menopause, elderly men do develop significant decreases in bioavailable (or non-sex hormone binding globulin-bound) testosterone and estrogen (E) levels. Moreover, these declining sex steroid and, in particular, declining bioavailable E levels correlate with bone loss in men. The evidence for decreases in bioavailable E levels with aging in both women and men (albeit through different mechanisms) thus provides the conceptual framework for our central hypothesis that E deficiency leads to increased shunting of bone marrow stromal cells towards the adipocyte lineage at the expense of the osteoblast lineage. We will test our hypothesis using basic, in vitro mechanistic studies as well as in vivo mouse and human models within the context of our three Specific Aims: Aim 1: Use the bipotential mouse bone marrow stromal cell line, ST2, as well as mouse bone marrow mesenchymal cell cultures to examine the molecular mechanisms by which E inhibits adipogenesis and enhances osteoblast development; Aim 2: Use aging mice to define the relative contributions of aging vs. E deficiency towards the observed age- related increase in bone marrow adipogenesis. As part of these studies, use the findings from Aim 1 to select the target genes to be evaluated in vivo for changes with age and for modulation of these changes by E; and Aim 3: Assess, in postmenopausal women, the relative contributions of aging vs. E deficiency towards the increased bone marrow adipogenesis seen in these women, as well as the potential underlying mechanisms for this. Collectively, the proposed studies address key questions posed by RFA-AG-06-003 ("The Adipogenic Phenotype in Aging Musculoskeletal Tissues"), as they focus on the mechanisms that control differentiation of mesencymal stem cells down the osteoblastic or adipocytic lineages and test the plausible hypothesis that, like age-related bone loss, age-related bone marrow adipocyte accumulation is driven, at least in part, by declining bioavailable E levels. These studies are also of considerable clinical significance, since understanding the pathways used by E in limiting adipogenesis and enhancing osteoblast development could lead to new approaches to prevent the relative deficit in osteoblasts with aging, which would have significant implications for the prevention and treatment of age-related bone loss. [unreadable] [unreadable] [unreadable]